Method for diagnosing cancer by detecting the methylation of transitional zones

ABSTRACT

The present invention relates to a method for diagnosing cancer and predicting metastasis or prognosis by measuring the methylation of transitional zones and a primer for detecting the methylation. According to the present invention, a novel transitional zone is understood and a primer for detecting the methylation of the zone is provided, indicating that the present invention contributes to increase accuracy and liability of cancer prediction by measuring the methylation of transitional zones and chromosomal loss at the same time.

TECHNICAL FIELD

The present invention relates to a method for diagnosing cancer bydetecting the methylation of transitional zones, confirming themetastasis and prognosis and a primer for detecting methylation.

BACKGROUND ART

Cancer has been acknowledged as a genetic disorder caused by mutation ofa gene. The amino acid sequence and the functions of a protein aredetermined by the nucleotide sequence of DNA. However, the expression ofa protein is affected by DNA methylation. That is, the functions andexpression of a specific gene depend on the nucleotide sequence and DNAmethylation. In tumor tissues, those genetic and epigenetic changes aregenerally observed. Therefore, by detecting such genetic and epigeneticchanges in tumor tissues a cause of a specific cancer can be explained,providing advantageous information for studies on the prevention andtreatment of cancer.

The aspects of DNA methylation in tumor tissues are as follows:

1) Both DNA methylation and demethylation are observed in tumor tissues,

2) DNA methylation is observed in CpG islands adjacent genes, while DNAdemethylation is observed in repeated sequences, and

3) DNA methylation and demethylation play an independent role in thedevelopment and progress of cancer.

The details of the involvement of DNA methylation in cancer developmentand progress have not been clearly understood, yet. However, the variousaspects of DNA methylation involved in cell growth, differentiation andaging have been found out, making DNA methylation a major target ofstudy to understand various malignant phenotypes caused by geneticinstability.

Human genome is differentiated into each tissue by epigeneticreprogramming in the early stage of development. The epigeneticstructure is mostly established in the early stage of embryogenesis,which is largely divided into two parts; one is maintained all throughthe life-time and the other is the transitional zone which is variableaccording to the cell differentiation and repeated sequence of RNA.Retroelement, which takes more than 40% of human genome, is a repeatedsequence originated from endogenous retrovirus-like genetic element. Theretroelement induces methylation of itself and at the same time causesmethylation of neighboring DNA, indicating that the retroelement plays acrucial role in DNA methylation in the whole genome.

Chromosomal loss can be detected by a simple repeated sequence marker,reflecting the dosage reduction of genome. The dosage reduction bringsdosage compensation mechanism in action to keep the dosage of genome ineach individual. Thus, chromosomal loss induces demethylation of nucleicacid to compensate the dosage reduction, which seems how DNA methylationis involved in tumor progress. DNA demethylation shows similar geneexpression pattern to androgenic program represented by the epigenicactivation for cell invasion and transition to induce placentation inthe early stage of gestation and the degeneration and loss withparturition.

Therefore, the present inventors investigated DNA methylation inrelation to chromosomal loss in tumor tissues, and further confirmedthat DNA methylation can play an important role in diagnosing cancerbased on the findings by the inventors that DNA methylation is moreactively induced in transitional zones in between CPG island and its'neighboring retroelement rather than in CpG island.

1. Balmain A, Gray J, Ponder B. The genetics and genomics of cancer. NatGenet 2003; 33 S:238-24

2. Hong S J, Choi S W, Lee K H, Lee S, Min K O, Rhyu M G. Preoperativegenetic diagnosis of gastric carcinoma based on chromosomal loss andmicrosatellite instability. Int J Cancer. 2005 Jan. 10; 113(2):249-58.

3. Kim K M, Kwon M S, Hong S J, Min K O, Seo E J, Lee K Y et al. Geneticclassification of intestinal-type and diffuse-type gastric cancers basedon chromosomal loss and microsatellite instability. Virchows Arch 2003;443(4):491-50

4. Choi S W, Lee K J, Bae Y A, Min K O, Kwon M S, Kim K M et al. Geneticclassification of colorectal cancer based on chromosomal loss andmicrosatellite instability predicts survival. Clin Cancer Res 2002;8(7):2311-2322.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a method fordiagnosing cancer which enables the prediction of exact level of cancermetastasis and prognosis by examining tumor tissues with endoscopebefore surgical operation.

Technical Solution

To achieve the above object, the present invention provides a method fordiagnosing cancer and predicting metastasis and prognosis of cancer byinvestigating DNA methylation of transitional zones.

The present invention also provides a primer for investigating DNAmethylation of transitional zones.

The present invention further provides a simple repeated sequence markergroup capable of measuring loss of heterozygosity (LOH) and chromosomeinstability.

Hereinafter, the present invention is described in detail.

The present invention provides a method for diagnosing cancer andpredicting metastasis or prognosis by measuring the methylation oftransitional zones.

The newly found transitional zone is a variable region formed in betweenCpG island and its' neighboring retroelement located in thetranscription regulating region of the upper stream of a gene, andmethylation therein depends on the transcription density of the repeatedsequence. In addition to the transcription density dependentmethylation, this region exhibits different levels of variability andthe big difference in patterns between normal and tumor tissues,indicating that this zone can be utilized for diagnosing cancer.

The following standards are considered for distinguishing genes involvedin cancer progress: 1) the distance between a gene and a retroelement;2) the types of neighboring retroelements; 3) the retroelement density;4) the distance between genes; 5) relation between CpG island and aretroelement; and 6) the retroelement density in the inside of nucleus.Considering the above standards, 40 epigenes have been identified inrelation to diagnosis of cancer, which are divided according to theamount of nucleotide sequence of CpG island into two groups: oneincludes markers such as RABGEF1, STAG and CHGB prepared in nucleotidesequence rich CPG island and the other group includes TNFRSF14,SERPINB5, ANGPTL7, TFF2, BGLAP, MSLN, DDX53 and MAGEA2 prepared innucleotide sequence lacking CpG island. They can also be groupedaccording to the distance between nucleotide of CpG island and its'neighboring retroelement, which are VDR, ST14, CDKN2A, MYBPC2, RUNX3,RUNX2, MLH1, PTEN, etc, prepared in two or more regions includingproximal and distal.

If L1 or LTR is more dominant in the upper stream than Alu and if CpGisland nucleotide sequence is lacking in the starting point of a gene,the methylation of the transitional zone will be more efficient targetfor diagnosing cancer and predicting the progress thereof. If CpG islandnucleotide sequence is abundant in the starting point of a gene, markersprepared in between a retroelement and CpG island will be highlyeffective for the prediction.

Methylation can be measured by the conventional methods described inKorean Patent Publication No. 2004-001575, Korean Patent Publication No.2006-0026595 and Korean Patent Publication No. 2003-0069752.

The present invention also provides a primer for detecting themethylation of the transitional zone.

The primer can be effectively used for measuring the methylation levelof the transitional zone, which can be a set of a forward primer and areverse primer selected from a group consisting of those sequenceslisted in Table 1. This primer set can be applied to electrophoresis andmicroarray chip.

TABLE 1 CpG Amplicon nucleotide Forward Reverse size Tm sequence (5′to3′) (5′to 3′) (bp) (° C.) RABGEF1, U AAGTTGGAAGT CAAAATAAAAT 131 58 −0.2kb AGGGATTGAGT ACCACCCTAACA M GTCGGAAGTA GAAATAAAATA 128 58 GGGATTGAGCCCGCCCTAACG STAG1, U TTTTTAGGTTT ACCCTCAAATT 96 58 −0.4 kb TAGGGTTGGTTCCACAAAACA M TTTTTTAGGTT CTCGAATTTC 94 58 TTAGGGTCGGC CGCAAAACG CHGB, UGGGAGTAGGTTG CAAACCAAAAA 92 58 −0.3 kb AGGTATTTGAAGT ATAAACAACCA MGGAGTAGGTTG CGAACCAAAAA 92 58 AGGTATTCGAAGC ATAAACGACCG VDR, UTGGTAGTGATT CCTCACACCAA 130 58 −0.7 kb GTGGTTGATTAT TACCACAAAACA MGGTAGCGATCG CTCACGCCGAT 128 58 CGGTTGATTAC ACCACGAAACG +1.0 kb UGGTATTTTAGAT AAAACAACTTA 102 58 GTTTTGATTTTG TCCACCCACCAA M GGTATTTTAGACGAAACAACTTA 102 58 GTTTCGATTTCG TCCACCCGCCGA ST14, U GAAGGGGAGATCACCATCAC 136 58 −0.3 kb GATTGGAGGT CACAACAACA M GAAGGGGAGA TCACCATCAC136 58 GATCGGAGGC CACGACGACG −0.8 kb U GGAGATGTTT ACAACACATC 106 58TTAGGTGATT TCATCTTACA M GGAGACGTTT ACAACACGTC 106 58 TTAGGCGATCTCATCTTACG CDKN2A, U TGTTTATTTTTG AAAACTCAAA 129 56 −0.1 kb TTTTGTAGGTGACCATTCCAA M TGTTTATTTTC AAAACTCAAA 129 58 GTTTCGTAGGC ACCGTTCCGA −1.5kb U TTGGGATTAGG CTATAAAACCCT 130 58 TTTAGTTTTGG ATCAACTCACACT MTCGGGATTAGG AAACCCTATC 125 60 TTTAGTTTCG GACTCACGCT +0.8 kb UGTATTTTAGGAA TTTTCTCCCCA 101 58 GTTGTTGTTTGT ACCTCCCAACA M GTATTTTAGGAATTTTTCTCCCCA 102 60 GTCGTTGTTTGC ACCTCCCGACG PPARG, U GGTTAGGTTTTCCTAACTACAC 111 58 −0.2 kb GTGTTTTGATGT ACTCCATCCA M GGTTAGGTTTTCCTAACTACGC 111 58 GTGTTTTGACGC GCTCCATCCG MYBPC2, U TTTTTAATTTAAAAAACATCC 96 58 −1.2 kb GTGGGGTTTGT AACCAATCCA M TTTAATTTAG AAAAACGTCC94 60 CGGGGTTCGC AACCAATCCG −0.7 kb U TGTTTGTTTTG AACTCCAAAAT 125 58GGAAGAGTTGT TTCACACCCCA M TGTTCGTTTCG AACTCCGAAAT 125 60 GGAAGAGTCGCTTCGCGCCCCG RB1, U TGTAAAATG AAAACTCTC 116 58 −0.4 kb GATTGGGTGAAACCCCAC M TTGTAAAAC AAAACTCTC 116 58 GGATTGGGC GAACCCCGC RUNX3, UGATGTGTTGTAT TCCCCATTAA 97 56 −0.5 kb AGTTAATTGGT ACAACCTCCA MCGCGTCGTAT TCCCCGTTAA 95 58 AGTTAATCGGC ACGACCTCCG  1.7 kb UTGGGGTTAGATT ATAAAATCTTAC 107 56 TTTGTTGTTTTT AACCACCATCA M CGGGGTTAGATTATAAAATCTTAC 107 58 TTCGTTGTTTTC GACCACCGTCG +1.0 kb U GTTGTTTTAATGCAAAATAAAACA 147 59 GGAGTAGGGAT AAAACACCTCA M GTCGTTTTAATG GAAATAAAACG147 59 GGAGTAGGGAC AAAACGCCTCG PAX5, U GTAGGAGGATT CCTAAATTACA 115 59−1.0 kb TTTGGTTTGTT ACCCAACCTCA M AGGAGGATTT TAAATTACGA 111 59TTGGTTCGTC CCCAACCTCG MLH1, U TTTTGATGTAGATG ACCACCTCATCA 121 58 −0.6 kbTTTTATTAGGGTTG TAACTACCCACA T M ACGTAGACGTTT CCTCATCGTA 115 58TATTAGGGTCGC ACTACCCGCG −1.0 kb U GATTTTAGGATT AAACTACCTCCT 126 58GTTGATATGAGT AATCTTTATCCA M GATTTTAGGATT AACTACCTCCTA 125 58GTCGATATGAGC ATCTTTATCCG CDH1, U GGTGAATTTTTAG TCACAAATACTT 108 56  0 kbTTAATTAGTGGTAT TACAATTCCAACA M TGAATTTTTAGT ACAAATACTTTA 104 58TAATTAGCGGTAC CAATTCCGACG PTEN, U TTTTGTGTTTT AACCTCCCAAAA 124 58 −1.4kb GTAAGAATTGGT AAACACTATCA M TTTCGCGTTTT ACCTCCCGAAAA 123 60GTAAGAATCGGC AACGCTATCG −0.9 kb U TATTTTGTTGG AACTCCAAATCAA 96 58GTTTTTATGGT TTCACAACATCA M TATTTTGTCGG AAATCGATTC 90 58 GTTTTTACGGCGCGACGTCG KIAA1752, U TAATGGTTTTTGA CACAAACTATTAT 103 58 +0.4 kbGGATTGAGATTG CAACCAATCACA M TAATGGTTTTTG CACAAACTATTAT 103 62AGGATTGAGATC CAACCGATCACG FLJ43855, U TGGTTGTTATT CTAAACCACACT 88 56−1.1 kb TGGGGTGGTTG AAAAACAAACA M TGGTTGTTATT CTAAACCACACT 88 58TGGGGCGGTC AAAAACGAACG RUNX2, U GGTTTTGGAAAT AAACAACAAATC 96 58 −0.7 kbTGTATATGGTGT TCAAACCTACA M TTTCGGAAATT AACAACGAATC 93 58 GTATACGGCGCTCGAACCTACG −3.0 kb U TGTTTGAGTGTA TCTCTCAAATCC 123 59 TATGAGTGGATCACAAACAACCA M TGTTCGAGTGTA TCTCTCGAATCC 123 59 TATGAGTGGAC CACAAACGACCG−3.8 kb U AGGTTTAGTTA CCACTAAATA 113 59 GTTTTAGTTG CCCTAACAACA MAGGTTTAGTTA CCACTAAATA 113 59 GTTTTAGTCG CCCTAACAACG +1.6 kb UGTTTGAGGGTGG ACTACCCCAAA 127 59 GTGGTAGTTGT AAATCTAAATCA M GTTTGAGGGCGGACTACCCCGAAA 127 59 GTGGTAGTCGC AATCTAAATCG MUC8, U GGTAGGAGTTATAATACAAACACT 140 55  2.0 kb TAGGAGAGTATT CACCACCTAACCA M GGTAGGAGTTATAATACAAACGCT 140 60 TAGGAGAGTATC CACCGCCTAACCG ESR2, U TTTTTTTTAAGACTAAAAATAC 111 56  0.9 kb GATTTTGTGTGT ACATTCCACCA M TTTTTTTAAGGCCAACTAAAAAT 113 58 ATTTCGCGCGC ACACGTTCCACCG E2F4, U GTGGTTAGGAAACCCAACCT 106 58  0 kb ATGGAAGTG CCACCATCA M GGCGGTTAGG AACCCGACCT 10658 AACGGAAGC CCGCCATCG TNFRSF14, U GAATTTTGTGATT CTCTAAACAAAC 115 58−0.6 kb TATGTGATGATG ACAAACAATACA M GAATTTCGTGAT CTCTAAACAAAC 115 58TTACGTGACGAC ACAAACGATACG SERPINB5, U GAATATTTTATTT AAAAAACCTC 111 56−0.3 kb TTTGGTTTTGTG CAACATATTCA M TTATTTTTC AAAAAACCTC 104 54 GGTTTTGCGCAACATATTCG ANGPTL7, U GTAATAGTAAGT CCTACAAAAAT 120 58 +0.5 kbGTATGGAGTTGT CTAAATAACCA M GTAATAGTAAGC CCTACGAAAAT 120 58 GTATGGAGTCGCCTAAATAACCG TFF2, U GGTAGTTGTGT CACATAACCA 130 56  0.2 kb TTTGTGTAGGTATTTTCCACA M GGTAGTTGTGT CACGTAACCG 130 62 TTTGTGTAGGC ATTTTCCACG BGLAP,U AGGGTAGGGT AATACCTCAC 86 58  0.5 kb TTGAGTTGTT AATACCCCCA M AGGGTAGGGTAATACCTCGC 86 58 TTGAGTCGTC AATACCCCCG MSLN, U GGAGAGATTAGA CATAAACTCTTA103 55  0.8 kb GATGATTGTTGT TCCCCAATACA M GGAGAGATTAGA CGTAAACTCTTA 10360 GATGATCGTCGC TCCCCAATACG DDX53, U TGGTTTTTGGG CAAATCTACAA 105 57  0kb GTAATTTTTGT CCTATTTCCCA M TTTTATACGAT CAAATCTACGA 136 58 TCGGAATTCGACCCTATTTCCCG MAGEA2, U GTTAGGTTGT CCAAAAAAAT 92 59 −0.1 kb TGTTTAGGGTCACAAACCCA M GCGTTTGTTTT AAATCACGAACC 108 61 TTTTCGTCGAC CGAATATAACG

The present invention further provides a diagnostic kit for cancercontaining the primer applicable for the detection of the methylation ofthe transitional zone.

The primers included in the kit can be every sequence that can beamplified by binding complementarily to the sequence of the transitionalzone of RABGEF1, STAG, CHGB, TNFRSF14, SERPINB5, ANGPTL7, TFF2, BGLAP,MSLN, DDX53, MAGEA2, VDR, ST14, CDKN2A, MYBPC2, RUNX3, RUNX2, MLH1 orPTEN gene. And, a set of a forward and a reverse primer selected from agroup consisting of those sequences listed in Table 1 are morepreferred. The construction of such primers can be accomplished by theconventional method well known to those in the art and a PCR productamplified by using the primer using the diagnostic kit of the inventioncan be confirmed by RCR machine generally used by those in the art.

The present invention also provides a simple repeated marker group usedfor measuring the loss of heterozygosity (LOH) and chromosomeinstability.

The present invention provides 40 simple repeated sequence markers whichare useful for selecting cancer related chromosomes with frequentdeletions and have confirmed that these markers are very useful forclassifying those chromosomal variations according to the level of theloss of heterozygosity (LOH) into 4 groups: primary, LOH-L (low), LOH-H(high) chromosomal loss and microsatellite instability (MSI). Thegenetic variations can be divided into high-risk genotypes (LOH-H andLOH-B) and low risk genotypes (LOH-L and MIS), which are crucial factorsfor the prediction of survival rate of phase 2 and phase 3 stomachcancer patients (FIG. 4, FIG. 5 and FIG. 6).

By detecting the repeated sequence instability and chromosomal loss,which are two most peculiar pathological characteristics of stomachcancer, genotypes are classified to predict metastasis and recurrence.

The above marker group is a pair of a forward primer and a reverseprimer selected from a group consisting of those sequences listed inTable 2.

TABLE 2 Amplicon Hetero chr Marker Forward Reverse Tm Size (%)  3D3S1597 AATACACACAA CCTTTTTTTCA 56  80-100 80 ATGTCTCTCCC GTGGTATGC  3D3S1552 ATTCCATACT GCAAATGCC 56 140-160 62 GTAGGGAGTGT ATGCTGTA  3D3S1312 CTTCTCACTG GGCTCCCC 56 148~158 60 CATATGACTC AGGGTAAG  3 D3S1478GATGAAACTG CTGCCAGTAAT 62 109-140 79 TGATAGCACC GTAAATCTCC  3 D3S1619GTCCTGCAA TTGCTAGGAT 59 161-171 60 GACTCATTG GGTTGTTTTC  4 D4S1609TCTGAAAAT CATCATTACT 59 163-177 67 GCCCTTGACC GCTGGGATGC  4 D4S2946GTCAAGAGG ACCTGTCTGA 59 104-126 72 GCTGATTCTG ACTTGCGTG  4 D4S174GCAGTTCAAA CATTCCTAGA 54 114~134 77 GATGAAAGTG TGGGTAAAGC  4 D4S391CACATAACTT ACTGTTGTCA 59 164-185 86 CCCTTGCTGG AATCAGGCTC  4 D4S230GGTAAATAGG TTAGGATGCT 56 170-196 0 GAAAATGACA GACTTCACCA  5 D5S519CTACTACCAG ATCTGCAGTG 59 114-128 83 CAGCATTCTC TGAGGCAATG  5 D5S346ACTCACTCTAGT AGCAGATAAGAC 56  96-135 83 GATAAATCGGG AGTATTACTAGTT  5D5S409 GGGATGAAGT TAGGATGGCA 59 138-154 69 GTGGATAAAC GTGCTCTTAG  5D5S349 ATTTGGTTTCCA TTACACCCACC 62 140-158 72 TAGAATCTGAGA AGATTAAGCG  5D5S422 TTAATTGATCTG CAGAGCAAGGTC 59 113-134 85 GGCTGGAGAACC CTGTCTGAAAAT 8 D8S261 TGCCACTGTC TATGGCCCAG 59 128-144 41 TTGAAAATCC CAATGTGTAT  8D8S262 AGCTCAAAAG GGCAACAAAG 59 114-128 0 CGAAGGTGAT TGAGATCCTG  8D8S503 CGTTTGGAAAT TCGCTCAGAA 56 107-120 45 TGTCATTACC ACAAACCAA  8D8S552 AGGATTGTAA GGGACTTTTT 56 168-182 79 TTTCCTTGC GAAGGTTTG  8 D8S277GATTTGTCCT ACATGTTATGTT 56 120-140 74 CATGCAGTGT TGAGAGGTCTG  9 D9S157AGCAAGGCAA TGGGGATGCCCA 59 113-149 76 GCCACATTTC GATAACTATATC  9 D9S200GCATTTCACAGG CCTCTCTGC 59 107-127 68 AAATAATCTAAGG ATGCCCCAG  9 D9S270AGGTGTAGTCC GATGTGACTGCTG 59  87-101 71 TTCTGGAATTT TTAAAACTAGAG  9D9S199 ACACATTCATA GGGGAAAGCA 56 144-164 77 CCATAGCAGAGG TTCAGACTTT  9D9S288 AGCAACCT AATCATCCA 62 124-140 72 CAACAGGG GAAAGGCCA 13 D13S267GGCCTGAAA TCCCACCAT 56 148-162 69 GGTATCCTC AAGCACAAG 13 D13S263CCTGGCCTGTTA CCCAGTCTTGG 56 145-165 0 GTTTTTATTGTTA GTATGTTTTTA 13D13S135 CCCTGTCTTCTA CGGTTCTCAA 59 168-174 75 CTTCCTGTATGC CCAGGAGAAA 13D13S286 TGATTGTATG TAGAGTGCAG 56  80-90 0 CATGCCTGTG TGTCCAAACG 13D13S118 TATAACTTGT CCACAGACAT 56 160-174 0 GTGAGCACAG CAGAGTCCTT 17 TPS3AGGGATACTATT ACTGCCACTCC 62 103-135 90 CAGCCCGAGGTG TTGCCCCATTC 17D17S122 CAGAACCACAAA GGCCAGACAGA 62 153-165 0 ATGTCTTGCATTC CCAGGCTCTGC17 D17S796 CAATGGAACC AGTCCGATAA 62 144-174 82 AAATGTGGTC TGCCAGGATG 17D17S1358 CCTAATTACACA TAATATAAGACT 52 148-160 0 ATACTTTTGGGGAACAAACAAATG 17 D17S1566 TTACTGAGCTG CTCTTACCTTGC 56 170-200 77TAATTCCATGAT TGGTGAGATTG 18 D18S67 AAGGAGTAACT CCTGCACTTGA 56 113-129 82TGGGTTCCATC TGAGATAGGC 18 D18S57 TTCAGGGTCT AGAAGGCATT 56  88-110 87TTTGAAGAGG AAATTTTGCA 18 D18S474 TGGGGTGTT TGGCTTTCAA 59 119-139 82TACCAGCATC TGTCAGAAGG 18 D18S70 AAGGCTGA GGAATGTCAAGA  0 111-126 0NCTCTACCG AGTACCTACCATA 18 D18S58 GCTCCCGG GCAGGAAATC 59 144-160 0CTGGTTTT GCAGGAACTT

The present invention also provides a diagnostic kit for cancercontaining the simple repeated sequence marker group.

The primers included in the kit are a pair of a forward primer and areverse primer selected from a group consisting of those sequenceslisted in Table 2. The construction of such primers can be accomplishedby the conventional method well known to those in the art and a PCRproduct amplified by using the primer in the diagnostic kit of theinvention can be confirmed by RCR machine generally used by those in theart.

The present inventors confirmed that methylation is increased with theincrease of chromosomal loss and decreased with the reduction ofchromosomal loss. Thus, investigation on methylation and measuring thelevel of LOH for the same lesion lead to the reduction of chances ofinaccuracy resulted from the discontinuity of chromosomal loss. Theresult from the genetic method provided by the present invention, whichwas the prediction of lymph node metastasis, was more accurate than thatfrom CT. The genetic diagnosis before surgical operation can provideinformation on the course of stomach cancer. Therefore, the method forgenetic diagnosis of the present invention is very useful for planningoperations and treatments.

DESCRIPTION OF DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a schematic diagram illustrating that methylation isdifferently induced in the transitional zone located in between CpGisland and a retroelement neighboring transcription starting pointaccording to the transcription density of repeated sequences in cells ortissues, indicating the tissue specific methylation with variations.Cancer progress would be predicted by detecting the epigenetic marker inthis variable region.

FIG. 2 is a block diagram illustrating that chromosome with LOH isquantified and the size of lesion is measured. Based on that,chromosomal loss is classified into LOH-H (high, 4˜8 loss) and LOH-L(low, 0˜3 loss) in the intestinal type and LOH-H (high, 4˜8 loss), LOH-L(low 2˜3 loss) and LOH-B (basal, 0˜1 loss) in the diffuse type. Thisclassification is helpful for the comparison of genotypes between genesobtained from the endoscopy tissue and genes obtained from the operationtissue. Based on the result of the genotype comparison and measurementof the size of the cancer lesion, cancer is diagnosed before surgicaloperation by using the LOH.

FIG. 3 is a photograph showing the results of PCR and electrophoresisexamining normal tissues and tumor tissues of each low risk group (case10) and high risk group (case 25) exhibiting chromosome instability byusing variable region epigenetic markers.

Case 10: low risk group (with chromosome instability) Case 25: high riskgroup (without chromosome instability but with LOH-H)

U: result of PCR using demethylation markers

M: result of PCR using methylation markers

%: degree of methylation

FIG. 4 is a photograph showing the results of PCR and electrophoresiswith normal and tumor tissues by using 40 simple repeated sequencemarkers located at 4p, 5q, 9p, 13q, 17p and 18q. * indicates the regionwith chromosomal loss confirmed by comparing with normal tissues andcase 25 shows LOH-H.

FIG. 5 is a set of graphs showing the survival curves obtained from 130stomach cancer patients having operation in phases 2 and 3. When thepatients are classified by genotype, the low risk group (LOH-L,chromosome instability) exhibits high survival rate, while the high riskgroup (LOH-H, LOH-B) exhibits low survival rate.

FIG. 6 is a set of graphs showing that chromosomal loss and themethylation of the transitional zone can be indexes for diagnosingcancer, by which high risk group (LOH-H, LOH-B) and low risk group(LOH-L, MSI) are clearly divided.

MODE FOR INVENTION

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Cancer Diagnosis Using PCR Markers Measuring the Methylationof the Transitional Zone <1-1> Micro-Dissection and DNA Extraction

A tumor tissue sample fixed in paraffin block was cut into 5 μm thick,and wax was eliminated by xylene, followed by hydration in ethanol. Thesample was stained with hematoxylin-eosin, which was then fixed on theslide glass.

With observing under the microscope, tumor tissues and normal tissues onthe slide glass were separated by using a needle. Each tissue obtainedfrom both normal and tumor region was put in lysis buffer, which stoodat 37° C. for 3 hours and then at 50° C. for 3 hours. The sample washeated before PCR to inactivate protease K.

<1-2> Bisulfite Modification and Methylation Specific PCR for ObtainingSamples for Analysis of Methylation Pattern

NaOH was added to the DNA obtained in Example <1-1>, which stood at 37°C. for 10 minutes. Hydroquinone and sodium bisulfite (pH 5.0) were addedthereto, followed by stirring. Mineral oil was loaded on the mixture,followed by reaction for 16 hours at 50° C. Purification was performedby using wizard DNA purification resin (Promega, USA), followed byelution. NaOH was added to the reaction mixture at the level of 0.3 M,followed by reaction at room temperature for 5 minutes, leading to thecompletion of modification. Ethanol precipitation was performed byadding ethanol in the presence of glycogen and sodium acetate, which wasstirred and stood overnight at −20° C. Centrifugation was performed for30 minutes for precipitation, followed by washing with 70% ethanol. Theprepared sample was used directly or stored at −20° C. for further use.

PCR reaction mixture included 1× PCR buffer, dNTPs, P³²-dTTP, primersand bisulfite-modified DNA or unmodified DNA. Hot-start was performed at95° C. for 5 minutes, to which Taq polymerase was added foramplification. Final extension was performed at 72° C. for 10 minutes.PCR product was loaded on polyacrylamide gel, followed byelectrophoresis. Radioluminograph scanner (BAS 2500, Fuji Photo Film,Japan) was used for observation. The level of methylation (%) wascalculated by using standard calibration curve and represented bypercentage. In FIG. 3, U indicates the result of amplification usingdemethylation markers, M indicates the result of amplification usingmethylation markers and % indicates the degree (level) of methylation.

FIG. 3 shows the results of PCR and electrophoresis examining normaltissues and tumor tissues of each low risk group (case 10) and high riskgroup (case 25) exhibiting chromosome instability by using variableregion epigenetic markers and markers prepared from the nucleotidesequence around CpG island. When the markers prepared from nucleotidesequence around CpG island were used, the methylation patterns of normaltissues and tumor tissues were not much different. However, when themarkers prepared from the transitional zone out of CpG island sequenceregion were used, low risk group (case 10) showed methylation but highrisk group (case 25) showed demethylation. The above results indicatethat the difference of the markers of CpG island sequence region was notsignificant according to normal or tumor tissues, but the markers of thetransitional zone is more sensitive to methylation, making thetransitional zone markers for better candidates for cancer diagnosis. Inhigh risk group (case 25), the methylation patterns of those markers ofCpG island such as P15 or RASSF1A were similar between normal tissuesand cancer tissues, while the methylation patterns of those markers suchas MLH1 or MAGEA2 of the transitional zone between normal and tumortissues were significantly different. In high risk group (case 25), themethylation of MLH1 (−0.6 kb) of CpG island sequence region andchromosomal loss were not significantly different between normal andtumor tissues, indicating that the MLH1 is a less preferable marker forcancer diagnosis. However the methylation of the transitional zone ofthe invention was significantly different between normal and tumortissues.

Example 2 Measurement of LOH (Loss of Heterozygosity) by Simple RepeatedSequence Markers

PCR and electrophoresis were performed with normal and tumor tissues(stomach cancer) by using 40 simple repeated sequence markers located at4p, 5q, 9p, 13q, 17p and 18q. As a result, at least 40% of homozygousmarkers (15 in total) exhibited high chromosome instability in case 10,while LOH-H chromosomal loss was detected in case 25.

Example 3 Comparison of Chromosomal Loss Between the Endoscopy Lesionand the Operation Lesion

As long as the endoscopy lesion can represent the total genotype, lossof heterozygosity (LOH) of the endoscopy lesion can be used for geneticdiagnosis before surgery. 91 pairs of the endoscopy lesion and theoperation lesion were compared and as a result 96% exhibited similargenotypes, suggesting that genetic diagnosis of the endoscopy lesion ispossible in stomach cancer patients before operation.

Example 4 Accuracy of the Prediction of the Operation Tissue Confirmedby Combinational Assay of LOH Level and the Size of a Lesion

From the multifocal study on 130 stomach cancer patients, it wasconfirmed that lymph node metastasis was frequent in high risk genotyperegardless of the size of a lesion. The lymph node metastasis was 83% inat least 5 cm lesion of low risk group. Intestinal invasion was frequentin larger lesion (>5 cm) than in smaller lesion (<5 cm) regardless ofgenotypes. Considering genotype of the endoscopy lesion together withthe size of a lesion, prediction for operation tissue can be accurateand precisely receiver operating characteristics area (Roc area) forlymph node metastasis and intestinal invasion were 0.815 and 0.685,respectively.

Example 5 Comparison of the Accuracy Between the Prediction of theOperation Tissue Made by Combinational Assay of the Level of LOH and theSize of a Lesion and the Result of Computer Tomography

Table 3 illustrates the examples in which the accuracy of combinationalassay of LOH and lesion size was compared with the result of computertomography. As a result, the prediction by the combinational assay ofLOH and lesion size was more accurate than the result of computertomography (intestinal invasion; ROC area=0.691 vs 0.548) or (lymph nodemetastasis; ROC area=0.691 vs 0.642).

TABLE 3 Pathology Genotype-size Computed tomography p value²Extraserosal No Yes No Yes invasion (%) No 38 (42) 21 (70) 17 (28) 23(46) 15 (37) Yes 53 (58)  9 (30) 44 (72) 27 (54) 26 (63) ROC area¹ 0.6910.548 0.076 Lymph node No Yes No Yes metastasis (%) No 36 (40) 25 (83)11 (18) 22 (61) 14 (39) Yes 55 (60)  5 (17) 50 (82) 18 (33) 37 (67) ROCarea¹ 0.802 0.642 0.021 ¹Area under the ROC curve ²Comparison of ROCareas between microsatellite genotype and computed tomography.

Example 6 Relation of Survival Rate with a Genotype Classified Accordingto LOH and Chromosome Instability

FIG. 5 is a set of graphs showing the survival curves obtained from 130stomach cancer patients having operation in phases 2 and 3. When thepatients were classified by the genotype, low risk groups (LOH-L,chromosome instability) showed high survival rate, while high riskgroups (LOH-H, LOH-B) showed low survival rate.

Example 7 Increasing the Accuracy of the Genetic Diagnosis by MeasuringBoth the Level of LOH and the Level of the Methylation of the VariableRegion

FIG. 6 is a set of graphs illustrating that the division of LOH intohigh, low, basal and chromosome instability was more clearly confirmedby measuring methylation. FIG. 6A and FIG. 6B are graphs illustratingthe decrease of methylation with the decrease of chromosomal loss andFIG. 6C and FIG. 6D are graphs illustrating the decrease of methylationwith the increase of chromosomal loss. In high and basal levels ofchromosomal loss, methylation was reduced, while in low level ofchromosomal loss and chromosome instability, methylation was increased.Using this difference of methylation according to the level ofchromosomal loss enhances the liability of the prediction of cancerdiagnosis. That is, methylation is closely related to the level of LOH,so that measuring methylation can reduce the inaccuracy caused by LOHdiscontinuity, suggesting that co-measuring the both LOH level andmethylation increases accuracy and liability to the prediction ofgenetic diagnosis.

INDUSTRIAL APPLICABILITY

The present invention provides a method for increasing the accuracy ofcancer diagnosis and prediction by 1) providing a marker group availablefor cancer diagnosis by measuring the methylation of transitional zones,2) providing a simple repeated sequence marker group in relation to LOH,and 3) co-measuring the methylation of transitional zones and the levelof LOH. Accordingly, the present invention provides a method forpre-surgery genetic diagnosis that is able to predict metastasis andprognosis with a small part of lesion, an endoscopy tissue, which willbe effectively used for planning the surgery and treatment for cancer.

[Sequence List Text]

Sequences represented by SEQ. ID. NO: 1˜NO: 160 are forward primers andreverse primers for 40 epigenetic markers of transitional zones whichare involved in cancer diagnosis. Sequences represented by SEQ. ID. NO:1˜NO: 4 are forward and reverse primers for RABGEF1, ˜0.2 kb, sequencesrepresented by SEQ. ID. NO: 5˜NO: 8 are forward and reverse primers forSTAG1, −0.4 kb, sequences represented by SEQ. ID. NO: 9˜NO: 12 areforward and reverse primers for CHGB, −0.3 kb, sequences represented bySEQ. ID. NO: 13˜NO: 16 are forward and reverse primers for VDR, −0.7 kb,sequences represented by SEQ. ID. NO: 17˜NO: 20 are forward and reverseprimers for VDR, +1.0 kb, sequences represented by SEQ. ID. NO: 21˜NO:24 are forward and reverse primers for ST14, −0.3 kb, sequencesrepresented by SEQ. ID. NO: 25˜NO: 28 are forward and reverse primersfor ST14, −0.8 kb, sequences represented by SEQ. ID. NO: 29˜NO: 32 areforward and reverse primers for CDKN2A, −0.1 kb, sequences representedby SEQ. ID. NO: 33˜NO: 36 are forward and reverse primers for CDKN2A,−1.5 kb, sequences represented by SEQ. ID. NO: 37˜NO: 40 are forward andreverse primers for CDKN2A, +0.8 kb, sequences represented by SEQ. ID.NO: 41˜NO: 44 are forward and reverse primers for PPARG, −0.2 kb,sequences represented by SEQ. ID. NO: 45˜NO: 48 are forward and reverseprimers for MYBPC2, −1.2 kb, sequences represented by SEQ. ID. NO:49˜NO: 52 are forward and reverse primers for MYBPC2, −0.7 kb, sequencesrepresented by SEQ. ID. NO: 53˜NO: 56 are forward and reverse primersfor RB1, −0.4 kb, sequences represented by SEQ. ID. NO: 57˜NO: 60 areforward and reverse primers for RUNX3, −0.5 kb, sequences represented bySEQ. ID. NO: 61˜NO: 64 are forward and reverse primers for RUNX3, −1.7kb, sequences represented by SEQ. ID. NO: 65˜NO: 68 are forward andreverse primers for RUNX3, +1.0 kb, sequences represented by SEQ. ID.NO: 69˜NO: 72 are forward and reverse primers for PAX5, −1.0 kb,sequences represented by SEQ. ID. NO: 73˜NO: 76 are forward and reverseprimers for MLH1, −0.6 kb, sequences represented by SEQ. ID. NO: 77˜NO:80 are forward and reverse primers for MLH1, −1.0 kb, sequencesrepresented by SEQ. ID. NO: 81˜NO: 84 are forward and reverse primersfor CDH1, 0 kb, sequences represented by SEQ. ID. NO: 85˜NO: 88 areforward and reverse primers for PTEN, −1.4 kb, sequences represented bySEQ. ID. NO: 89˜NO: 92 are forward and reverse primers for −0.9 kb,sequences represented by SEQ. ID. NO: 93˜NO: 96 are forward and reverseprimers for KIAA1752, +0.4 kb, sequences represented by SEQ. ID. NO:97˜NO: 100 are forward and reverse primers for FLJ43855, −1.1 kb,sequences represented by SEQ. ID. NO: 101˜NO: 104 are forward andreverse primers for RUNX2, −0.7 kb, sequences represented by SEQ. ID.NO: 105˜NO: 108 are forward and reverse primers for RUNX2, −3.0 kb,sequences represented by SEQ. ID. NO: 109˜NO: 112 are forward andreverse primers for RUNX2, −3.8 kb, sequences represented by SEQ. ID.NO: 113˜NO: 116 are forward and reverse primers for RUNX2, +1.6 kb,sequences represented by SEQ. ID. NO: 117˜NO: 120 are forward andreverse primers for MUC8, +2.0 kb, sequences represented by SEQ. ID. NO:121˜NO: 124 are forward and reverse primers for ESR2, −0.9 kb, sequencesrepresented by SEQ. ID. NO: 125˜NO: 128 are forward and reverse primersfor E2F4, 0 kb, sequences represented by SEQ. ID. NO: 129˜NO: 132 areforward and reverse primers for TNFRSF14, −0.6 kb, sequences representedby SEQ. ID. NO: 133˜NO: 136 are forward and reverse primers forSERPINB5, −0.3 kb, sequences represented by SEQ. ID. NO: 137˜NO: 140 areforward and reverse primers for ANGPTL7, +0.5 kb, sequences representedby SEQ. ID. NO: 141˜NO: 144 are forward and reverse primers for TFF2,−0.2 kb, sequences represented by SEQ. ID. NO: 145˜NO: 148 are forwardand reverse primers for BGLAP, −0.5 kb, sequences represented by SEQ.ID. NO: 149˜NO: 152 are forward and reverse primers for MSLN, −0.8 kb,sequences represented by SEQ. ID. NO: 153˜NO: 156 are forward andreverse primers for DDX53, 0 kb, and sequences represented by SEQ. ID.NO: 157˜NO: 160 are forward and reverse primers for MAGEA2, −0.1 kb.

Sequences represented by SEQ. ID. NO: 161˜NO: 240 are simple repeatedsequence markers available for measuring LOH and chromosome instability.SEQ. ID. NO: 161 is a forward primer and SEQ. ID. NO: 162 is a reverseprimer for D3S1597, SEQ. ID. NO: 163 is a forward primer and SEQ. ID.NO: 164 is a reverse primer for D3S1552, SEQ. ID. NO: 165 is a forwardprimer and SEQ. ID. NO: 166 is a reverse primer for D3S1312, SEQ. ID.NO: 167 is a forward primer and SEQ. ID. NO: 168 is a reverse primer forD3S1478, SEQ. ID. NO: 169 is a forward primer and SEQ. ID. NO: 170 is areverse primer for D3S1619, SEQ. ID. NO: 171 is a forward primer andSEQ. ID. NO: 172 is a reverse primer for D4S1609, SEQ. ID. NO: 173 is aforward primer and SEQ. ID. NO: 174 is a reverse primer for D4S2946,SEQ. ID. NO: 175 is a forward primer and SEQ. ID. NO: 176 is a reverseprimer for D4S174, SEQ. ID. NO: 177 is a forward primer and SEQ. ID. NO:178 is a reverse primer for D4S391, SEQ. ID. NO: 179 is a forward primerand SEQ. ID. NO: 180 is a reverse primer for D4S230, SEQ. ID. NO: 181 isa forward primer and SEQ. ID. NO: 182 is a reverse primer for D5S519,SEQ. ID. NO: 183 is a forward primer and SEQ. ID. NO: 184 is a reverseprimer for D5S346, SEQ. ID. NO: 185 is a forward primer and SEQ. ID. NO:186 is a reverse primer for D5S409, SEQ. ID. NO: 187 is a forward primerand SEQ. ID. NO: 188 is a reverse primer for D5S349, SEQ. ID. NO: 189 isa forward primer and SEQ. ID. NO: 190 is a reverse primer for D5S422,SEQ. ID. NO: 191 is a forward primer and SEQ. ID. NO: 192 is a reverseprimer for D8S261, SEQ. ID. NO: 193 is a forward primer and SEQ. ID. NO:194 is a reverse primer for D8S262, SEQ. ID. NO: 195 is a forward primerand SEQ. ID. NO: 196 is a reverse primer for D8S503, SEQ. ID. NO: 197 isa forward primer and SEQ. ID. NO: 198 is a reverse primer for D8S552,SEQ. ID. NO: 199 is a forward primer and SEQ. ID. NO: 200 is a reverseprimer for D8S277, SEQ. ID. NO: 201 is a forward primer and SEQ. ID. NO:202 is a reverse primer for D9S157, SEQ. ID. NO: 203 is a forward primerand SEQ. ID. NO: 204 is a reverse primer for D9S200, SEQ. ID. NO: 205 isa forward primer and SEQ. ID. NO: 206 is a reverse primer for D9S270,SEQ. ID. NO: 207 is a forward primer and SEQ. ID. NO: 208 is a reverseprimer for D9S199, SEQ. ID. NO: 209 is a forward primer and SEQ. ID. NO:210 is a reverse primer for D9S288, SEQ. ID. NO: 211 is a forward primerand SEQ. ID. NO: 212 is a reverse primer for D13S267, SEQ. ID. NO: 213is a forward primer and SEQ. ID. NO: 214 is a reverse primer forD13S263, SEQ. ID. NO: 215 is a forward primer and SEQ. ID. NO: 215 is areverse primer for D13S135, SEQ. ID. NO: 217 is a forward primer andSEQ. ID. NO: 218 is a reverse primer for D13S286, SEQ. ID. NO: 219 is aforward primer and SEQ. ID. NO: 220 is a reverse primer for D13S118,SEQ. ID. NO: 221 is a forward primer and SEQ. ID. NO: 222 is a reverseprimer for TP53, SEQ. ID. NO: 223 is a forward primer and SEQ. ID. NO:224 is a reverse primer for D17S122, SEQ. ID. NO: 225 is a forwardprimer and SEQ. ID. NO: 226 is a reverse primer for D17S796, SEQ. ID.NO: 227 is a forward primer and SEQ. ID. NO: 228 is a reverse primer forD17S1358, SEQ. ID. NO: 229 is a forward primer and SEQ. ID. NO: 230 is areverse primer for D17S1566, SEQ. ID. NO: 231 is a forward primer andSEQ. ID. NO: 232 is a reverse primer for D18S67, SEQ. ID. NO: 233 is aforward primer and SEQ. ID. NO: 234 is a reverse primer for D18S57, SEQ.ID. NO: 235 is a forward primer and SEQ. ID. NO: 236 is a reverse primerfor D18S474, SEQ. ID. NO: 237 is a forward primer and SEQ. ID. NO: 238is a reverse primer for D18S70, SEQ. ID. NO: 239 is a forward primer andSEQ. ID. NO: 240 is a reverse primer for D18S58.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A genetic marker for cancer diagnosis, the genetic marker containingone or more transitional zones selected from a group consisting ofRABGEF1 (RAB guanine nucleotide exchange factor 1), STAG (stromalantigen), CHGB (chromogranin B), TNFRSF14 (tumor necrosis factorreceptor superfamily 14), SERPINB5 (serine proteinase inhibitor, cladeB, member 5), ANGPTL7 (angiopoietin-like 7), TFF2 (trefoil factor 2),BGLAP (bone gamma-carboxyglutamate (gla) protein), MSLN (mesothelin),DDX53 (DEAD (SEQ ID NO: 241) box polypeptide 53), MAGEA2 (melanomaantigen family A), VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor),ST14 (suppression of tumorigenicity 14), CDKN2A (cyclin-dependent kinaseinhibitor 2A), MYBPC2 (myosin binding protein C, fast type), RUNX3(runt-related transcription factor 3), RUNX2 (runt-related transcriptionfactor 2), MLH1 (MutL DNA mismatch repair protein) and PTEN (Phosphataseand Tensin homolog deleted on chromosome Ten).
 2. A method for one orboth of diagnosing cancer and predicting metastasis or prognosis bymeasuring the DNA methylation of a tissue sample, the method comprising:preparing a tissue sample for measuring the DNA methylation of one ormore transitional zones of the tissue sample; and measuring the DNAmethylation of the one or more transitional zones.
 3. The methodaccording to claim 2, wherein the transitional zone is one or more zonesselected from a group consisting of RABGEF1, STAG, CHGB, TNFRSF14,SERPINB5, ANGPTL7, TFF2, BGLAP, MSLN, DDX53, MAGEA2, VDR, ST14, CDKN2A,MYBPC2, RUNX3, RUNX2, MLH1 and PTEN.
 4. A primer for detecting the DNAmethylation of one or more transitional zones.
 5. The primer accordingto claim 4, wherein the primer comprises a set of forward and reverseprimers selected from a group consisting of sequences represented bySEQ. ID. NO: 1˜NO:
 160. 6. A diagnostic kit for diagnosing cancer,wherein the diagnostic kit contains the primer of claim
 4. 7. A simplerepeated sequence marker group for measuring the loss of heterozygosity(LOH) and the level of chromosome instability.
 8. The marker groupaccording to claim 7, wherein the marker group comprises a set offorward and reverse primers selected from a group consisting ofsequences represented by SEQ. ID. NO: 161˜NO:
 240. 9. A diagnostic kitfor diagnosing cancer, wherein the diagnostic kit contains the markergroup of claim
 7. 10. The method according to claim 2, furthercomprising measuring the level of LOH.
 11. The diagnostic kit for canceraccording to claim 6, wherein the kit further comprises a simplerepeated sequence marker group available for measuring the loss ofheterozygosity (LOH) and the level of chromosome instability.
 12. Thegenetic marker of claim 1, wherein the cancer diagnosis comprisesdiagnosing stomach cancer.
 13. The genetic marker of claim 1, whereinthe genetic marker is used in the diagnosis of cancer.
 14. The methodaccording to claim 2, wherein the transitional zone comprises a regionformed in between a CpG island and a neighboring retroelement.
 15. Themethod according to claim 2, wherein the transitional zone ischaracterized by one or more of transcriptional density dependentmethylation, differing levels of variability, and differences inpatterns between normal and tumor tissues.
 16. The method according toclaim 2, wherein the cancer comprises stomach cancer.
 17. The methodaccording to clam 2, wherein the diagnosis or prediction is apreoperative diagnosis or prediction.
 18. The method according to claim17, wherein the diagnosis or prediction is made using anendoscopically-obtained tissue sample.
 19. The diagnostic kit of claim6, further comprising one or more DNA methylation reagents required toaffect detection of DNA methylated transitional zones.
 20. Thediagnostic kit of claim 9, further comprising one or more DNAmethylation reagents required to affect detection of DNA methylatedtransitional zones.